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How Breast Cancer Cells Evade Death

 

By: Carol A. Rouzer, VICB Communications
Published:  June 18, 2015

 

 

New studies highlight the importance of myeloid cell leukemia-1 (Mcl-1) for the survival and proliferation of triple negative breast cancer cells.

 

Cancer cells experience a high degree of genetic and metabolic stress. Exposure to radiation or chemotherapy increases that stress. Under these conditions, most normal cells undergo apoptosis or necrosis, but cancer cells have developed mechanisms to bypass these pathways that are intended to eliminate damaged cells. One mechanism by which cancer cells evade apoptosis is by altering the normal balance between the anti- and pro-apoptotic members of the Bcl-2 family of proteins. For example, under normal circumstances, stress leads to activation of the pro-apoptotic BH3-only proteins, such as Bim, Bad, and Noxa. These proteins bind to the multi-domain anti-apoptic proteins, including Mcl-1, Bcl-2, and Bcl-xL. This binding causes the anti-apoptotic proteins to dissociate from Bak and Bax, thereby freeing them to aggregate and form pores in the mitochondrial membrane. The result is release of cytochrome C from the mitochondria into the cytosol, an initial key step in the intrinsic apoptotic pathway (Figure 1). Many cancer cells defeat this pathway through gene amplification, transcriptional upregulation, or reduced degradation of the anti-apoptotic proteins. Potent small-molecule inhibitors have been developed against Bcl-2 and Bcl-xL, and these are highly effective at restoring apoptosis in cell lines that depend on those proteins for survival. Considerable effort is currently directed towards the discovery of Mcl-1 inhibitors as well. However, this therapeutic approach requires that the specific protein or proteins responsible for apoptosis evasion in a given cancer are correctly identified. Now, Vanderbilt Institute of Biology member Steve Fesik and his laboratory define the role of Mcl-1 in triple negative breast cancer [C. M. Goodwin, et al. (2015) Cell Death Differ., Published online June 5, doi: 10.1038/cdd.2015.73].

 

 

 


Figure 1. Examples of how Bcl-2 proteins interact to control programmed cell death (apoptosis). A. An apoptotic signal activates the proapoptotic protein Bak, causing it to bind to the outer mitochondrial membrane. If it oligomerizes with other Bak proteins, it will form a pore in the membrane allowing release of contents, including the protein cytochrome C (CC), which is an early step in the apoptotic pathway. B. The antiapoptotic protein Mcl-1 binds to Bak and prevents its self-oligomerization. C. Other proteins, such as Bim can bind to Mcl-1, causing it to release Bak. D. No longer bound to Mcl-1, Bak self-oligomerizes, causing the release of cytchrome C.

 


Breast cancer is the most frequent form of cancer among women in the United States, and second only to lung cancer as a cause of cancer-related deaths. Approximately 15% of breast cancers are of the triple negative subtype (TNBC), meaning that the tumors do not express the estrogen, progesterone, or human epidermal growth factor 2 (Her2/neu) receptors. The absence of these receptors renders the tumors resistant to many of the most effective treatments for breast cancer, and patients suffering from TNBC have a poor prognosis. Overexpression of Mcl-1 and/or Bcl-xL is frequently found in breast cancer and has been associated with resistance to therapy. This led the Fesik lab investigators to hypothesize that one or both of these anti-apoptotic Bcl-2 family proteins may play a role in TNBC.

 

To test their hypothesis, the investigators first used siRNA to silence expression of Mcl-1 or Bcl-xL in 17 publicly available TNBC cell lines. Mcl-1 knockdown reduced cell viability by 60% or more in seven of the lines, suggesting that these lines were highly dependent on Mcl-1 for growth and survival. Four of the 17 lines exhibited 40-60% reduction in viability in response to Mcl-1 knockdown, indicating a partial dependence on Mcl-1. The remainder were unaffected by Mcl-1 knockdown. Mcl-1 knockdown was correlated with activation of caspase 3/7 and surface expression of Annexin V, two hallmarks of apoptosis, in those cell lines that exhibited loss of viability, confirming that the cells required high expression of Mcl-1 to avoid apoptosis. Although only two cell lines were sensitive to Bcl-xL knockdown, simultaneous knockdown of both Bcl-xL and Mcl-1 significantly reduced cell viability in most of the lines.

 

The siRNA results suggested a strong role for Mcl-1 in apoptosis resistance of some TNBCs and a lesser role for Bcl-xL. To further explore these relationships, the investigators used synthetic peptides that specifically inhibit one or more of the anti-apoptotic Bcl-2 proteins. They introduced the peptides into the cells by membrane permeabilization and measured the extent of mitochondrial depolarization introduced through their inhibition of Mcl-1, Bcl-xL, or Bcl-2. Binding of the peptide to its target protein prevents it from interacting with Bak or Bax. The investigators found that Bim-BH3, a peptide that binds to all of the anti-apoptotic Bcl-2 proteins, caused mitochondrial depolarization in all the TNBC cell lines tested. MS-1, a peptide that targets only Mcl-1, caused depolarization only in the cell lines previously found to be Mcl-1-dependent by siRNA. Bad-BH3, a peptide that targets Bcl-2, Bcl-xL, and Bcl-w, caused depolarization of some cell lines, but was not as effective as MS-1, suggesting that Bcl-xL plays a secondary role to Mcl-1 to prevent apoptosis in these cell lines. Consistent with the siRNA results, exposure of cells to MS-1 and Bad-BH3 produced additive, or even synergistic results.

 

Although the results indicated that TNBC cells were generally not highly dependent on Bcl-xL, they also demonstrated that suppression of both Bcl-xL and Mcl-1 was highly effective at inhibiting growth and survival of most TNBC cell lines. Consistently, the investigators found that TNBC cells were not very sensitive to WEHI-539, a Bcl-xL-selective small molecule inhibitor. However, this inhibitor became quite effective in the presence of siRNA-mediated Mcl-1 knockdown. Similar results were found for ABT-263, which blocks the activity of both Bcl-2 and Bcl-xL but not for the Bcl-2-selective inhibitor ABT-199, which was ineffective against TNBC cell lines whether or not Mcl-1 expression was suppressed.

 

Since Mcl-1 works by interacting with other Bcl-2 family members, the investigators hypothesized that the dependency of TNBC cell lines on Mcl-1 could be attributable to the activity of other, pro-apoptotic proteins in the pathway. They found, however, that expression levels of no single family member strongly correlated with Mcl-1-dependency, although high expression of pro-apoptotic Bak and Bax did weakly correlate with Mcl-1 dependency. In contrast, multiple linear regression analysis provided an equation that correlated Mcl-1-dependency to the levels of expression of Mcl-1 itself plus Bcl-xL, Noxa, Bim, and Bak, as determined by immunoblot analysis. Moreover, this equation showed utility in predicting sensitivity in lung cancer.

 

The results confirm that a subset of TNBC tumors is highly dependent on Mcl-1 for proliferation and survival. All of these tumors fall into the basal A subgroup of TNBC. The findings suggest that this group of TNBC tumors would likely respond well to therapy designed to suppress the activity of Mcl-1 and that therapy designed to simultaneously block both Mcl-1 and Bcl-xL would be particularly effective. The Fesik laboratory is actively seeking an inhibitor of Mcl-1, and approval of new drugs that target these Bcl-2 family members could be an important step forward for some TNBC patients, as well as sufferers of many other forms of cancer that depend on these proteins for survival.

 


 


 

 

 

 

 

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